Active-electrode integrated biosensor array and methods for use thereof

ABSTRACT

A method and device for performing DNA sequencing and extracting structural information from unknown nucleic acid strands. The device includes a microwell structure, where identical DNA strands are immobilized within the microwell structure on a surface of a micro-bead, an active electrode or a porous polymer. The device further includes a CMOS-integrated semiconductor integrated circuit, where the CMOS-integrated semiconductor integrated circuit includes metal layers on a silicon substrate, where the metal layers form an active electrode biosensor. In addition, a sensing electrode is formed by creating openings in a passivation layer of the CMOS-integrated semiconductor integrated circuit to hold a single bead, on which the DNA strands are immobilized.

TECHNICAL FIELD

The present invention relates generally to biosensors andbioelectronics, and more particularly to a type of electro-analyticalbiosensor, referred to herein as the “active-electrode” biosensor, thatis compatible with Very Large Scale Integration (VLSI) manufacturingprocesses and is used in genomics and proteomics applications.

BACKGROUND

Biosensors are devices that use biochemical reactions to identify anddetect various molecules and biochemical analytes. Biosensors are widelyused in different life-science applications, ranging from environmentalmonitoring and basic life science research to Point-of-Care (PoC)in-vitro molecular diagnostics. Biosensors are known to be verysensitive and also extremely versatile in terms of detection as they candetect a small number of almost any kind of analyte, once a properrecognition molecule is identified. Example analytes that have beendetected using biosensors include DNA and RNA strands, proteins,metabolites, toxins, micro-organisms, and even explosives molecules.

All biosensors, independent of the analyte they are trying to detect,include two key building blocks. One is the molecular recognition layerwhich is responsible for identifying and/or interacting with and/orreacting with and/or capturing the specific target analyte from thesample. The other is the sensor apparatus that detects and/or quantifiesthe interactions of the recognition layer with the analyte and providesa measurable output signal, generally in the form of an electricalsignal. The molecular recognition layer typically comprises of carefullyengineered and surface-assembled bio-molecules in the form of spotted orsynthesized DNA oligonucleotides, aptamers, and antibodies attached tosolid substrates, such as glass slides, micro-beads, electrodes,semiconductor materials, or dense polymers while the sensor includesoptical-, MEMS- and/or electronics-based transducers connected to alow-noise detection circuit.

So far, there have been many detection methods that have been adopted inbiosensor systems. A detection method is defined as the specific type ofphysiochemical mechanism designed into the molecular recognition layer,analytes, and the interaction environments that make the identificationof the specific target analytes possible by the sensor. The most widelyused detection methods are different classes of optical (e.g.,fluorescence, bioluminescence) and electro-analytical (e.g.,potentiometric, amperometric, impedimetric). It is also common toclassify biosensors based on their detection method. For example, inbioluminescence-based biosensors, the interaction of the analyte andprobes results in a bioluminescence phenomenon which is detected by aspecific sensor with a transducer sensitive to bioluminescence signals.

Electro-analytical biosensors detect analytes by monitoring differentelectronic changes in electrode-electrolyte transducers that arespecifically interfaced with a recognition layer. For instance, inamperometric biosensors, low-frequency Faradaic reduction-oxidation(redox) currents are used as an indicator for analyte interactions withthe recognition layer, whereas in impedimetric biosensors, the changesin the electrode-electrolyte impedance induced by the captured analyteare used as an indicator of analyte interactions with the recognitionlayer.

Unfortunately, the existing state-of-the-art electro-analyticalbiosensors are not compatible with semiconductor Very Large ScaleIntegration (VLSI) manufacturing processes thereby not being able totake advantage of the VLSI processes (e.g., highest level ofintegration, miniaturization, cost-efficiency, and robustness).

BRIEF SUMMARY

As discussed above, the existing state-of-the-art electro-analyticalbiosensors are not compatible with semiconductor Very Large ScaleIntegration (VLSI) manufacturing processes thereby not being able totake advantage of the VLSI processes (e.g., highest level ofintegration, miniaturization, cost-efficiency, and robustness). Theprinciples of the present invention address this impediment.

In view of the limitations of biosensors currently available, there is aneed for improved biosensors and methods for use thereof in fields, suchas nucleic acid detection, nucleic acid sequencing, proteomics,forensics, in-vitro diagnostics, medicine, and the like. Neededimprovements include reducing the cost, increasing the throughput,and/or decreasing the size of biosensors instrument; in order to advancethe field of personalized medicine for example.

Provided herein are Complementary Metal Oxide Semiconductor (CMOS)biological sensors (“biosensors”) fabricated using Very Large ScaleIntegration (VLSI) manufacturing processes in various applications, suchas, for example, nucleic acid sequencing, proteomics, and forensics.CMOS biosensors described in various embodiments of the presentinvention can be used in a variety of genomics and proteomicsapplications. In particular, they can be used in nucleic acidsequencing, such as deoxyribonucleic acid (DNA) sequencing, ribonucleicacid (RNA) sequencing, and forensics analysis, such as short tandemrepeat (STR) analysis.

In one embodiment of the present invention, a biosensor comprises anelectrode disposed adjacent to a fluid layer having a charged speciestherein. The biosensor system further comprises an interface of theelectrode and the fluid layer characterized by a first capacitance,where the fluid layer is characterized by an impedance. Furthermore, thebiosensor system comprises detection circuitry operatively coupled tothe electrode, where the detection circuitry comprises an operationalamplifier and a capacitor, where the capacitor is in a parallelconfiguration with respect to the operational amplifier, where thedetection circuitry is configured to detect the charged species in thefluid layer, where the capacitor comprises a second capacitance and thedetection circuitry having a potential output that is a function of atleast one of (i) an induced potential within the fluid layer, (ii) thefirst capacitance and (iii) the second capacitance.

In another embodiment of the present invention, a biosensor comprises areaction chamber. The biosensor further comprises a sensing electrodeadjacent to the reaction chamber. Furthermore, the biosensor comprises astack of metal layers disposed adjacent to the sensing electrode, wherethe stack of metal layers comprises one or more metal layers separatedby an insulating material. In addition, the biosensor comprisesdetection circuitry adjacent to the stack of metal layers.

In another embodiment of the present invention, a method for detectingDNA polymerization using active electrode biosensors comprisesimmobilizing a plurality of primed DNA molecules in a reaction chamber.The method further comprises interfacing an active-electrode biosensorsystem to the plurality of primed DNA molecules. Furthermore, the methodcomprises introducing nucleotides of DNA into the reaction chamber inthe presence of a DNA polymerase enzyme. Additionally, the methodcomprises measuring an output voltage of the active-electrode biosensorsystem. The method further comprises estimating an ionic current usingthe measured output voltage. In addition, the method comprisesidentifying an occurrence and amount of DNA polymerization eventsassociated with the immobilized DNA using the estimated ionic current.

In another embodiment of the present invention, a method for performingparallel DNA sequencing comprises creating an immobilized primed DNAarray on a surface of a CMOS-integrated semiconductor integrated circuitwhere DNA strands are located in distinct coordinates within the DNAarray. The method further comprises building an array of activeelectrode biosensors in the CMOS-integrated semiconductor integratedcircuit. Furthermore, the method comprises establishing a reactionchamber which contains aqueous solutions on top of the CMOS-integratedsemiconductor integrated circuit where the DNA array is located.Additionally, the method comprises establishing a fluidicsingle-directional flow-through system which enables controlledinjection and removal of different aqueous from the reaction chamber. Inaddition, the method comprises establishing a device to extract, readand process an output of each integrated active electrode biosensor inthe array of active electrode biosensors.

In another embodiment of the present invention, a device forimmobilizing primed DNA strands comprises a microwell structure, whereidentical DNA strands are immobilized within the microwell structure ona surface of one of the following: a micro-bead, an active electrode anda porous polymer. The device further comprises a CMOS-integratedsemiconductor integrated circuit, where the microwell structure isplaced on top of the CMOS-integrated semiconductor integrated circuit.The CMOS-integrated semiconductor integrated circuit comprises metallayers on a silicon substrate, where the metal layers form an activeelectrode biosensor.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The novel features of the present invention are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of the present invention will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the invention areutilized, and the accompanying drawings of which:

FIG. 1 illustrates an active electrode circuit model in accordance withan embodiment of the present invention;

FIG. 2 illustrates an active electrode system using a switch capacitoramplifier sensor in accordance with an embodiment of the presentinvention;

FIG. 3A illustrates the correlated double sampling method to suppressthe low frequency noise and offset of the amplifier within theactive-electrode sensor in accordance with an embodiment of the presentinvention;

FIG. 3B is a timing diagram of the circuit of FIG. 3A in accordance withan embodiment of the present invention;

FIG. 4 illustrates using active electrode sensors to measure C_(D) inaccordance with an embodiment of the present invention;

FIG. 5 illustrates using active electrode sensors to measure the ioniccurrents within the electrolyte in accordance with an embodiment of thepresent invention;

FIG. 6 illustrates hybrid electro-analysis using an active electrodesensor in accordance with an embodiment of the present invention;

FIG. 7 is a table illustrating the absorbed ions and net free chargegenerated during a single nucleotide incorporation at different pHlevels in accordance with an embodiment of the present invention;

FIGS. 8A-8C illustrate different embodiments for immobilizing identicalDNA in proximity of an active electrode in accordance with an embodimentof the present invention;

FIGS. 9A-9B illustrate examples of CMOS-integrated sensing electrodes inaccordance with an embodiment of the present invention;

FIGS. 10A-10C illustrate an integrated active electrode DNA sequencingbiochip and the basic layer structure of its pixels in accordance withan embodiment of the present invention;

FIG. 11A illustrates the general architecture of the CMOS-integratedactive-electrode biosensor pixel for DNA sequencing in accordance withan embodiment of the present invention;

FIG. 11B is a timing diagram of in-pixel and out-of-pixel in accordancewith an embodiment of the present invention;

FIG. 12 is a transistor-level schematic of a signal chain in accordancewith an embodiment of the present invention;

FIGS. 13A-13C are graphs illustrating electrical detection performancesin accordance with an embodiment of the present invention;

FIGS. 14A-14C illustrate interface capacitance measurements versussolution pH in accordance with an embodiment of the present invention;

FIG. 15 illustrates DNA polymerization detection in accordance with anembodiment of the present invention; and

FIG. 16 is a micrograph of the active-electrode CMOS biochip for DNAsequencing in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION

Incorporation by Reference

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Principles of the Present Invention

While various embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions may occur to those skilled in theart without departing from the present invention. It should beunderstood that various alternatives to the embodiments of the presentinvention described herein may be employed in practicing the invention.

The principles of the preset invention relate to electro-analyticalbiosensors. Generally speaking, electro-analytical biosensors thatdetect analytes by monitoring different electronic changes inelectrode-electrolyte transducers that are specifically interfaced withthe recognition layers. For instance, in amperometric biosensors,low-frequency Faradaic reduction-oxidation currents are used as anindicator for analyte interactions with the recognition layer, whereasin impedimetric biosensors, the changes in the electrode-electrolyteimpedance induced by the captured analyte are used as an indicator ofanalyte interactions with the recognition layer.

It is noted that depending on the exact electrical characteristic thatis being probed (or monitored) in electro-analytical biosensors,different instrumentation techniques and electrode configurations arerequired. The principles of the present invention described hereindescribe a specific type of electro-analytical biosensor, called anactive-electrode biosensor, and the methods by which one can createhigh-performance and highly-parallel DNA sequencing platforms employingthis biosensor.

One of the key advantages of active-electrode biosensors is that theyare compatible with semiconductor Very Large Scale Integration (VLSI)manufacturing processes in general, and ComplementaryMetal-Oxide-Semiconductor (CMOS) Integrated Circuits (ICs) inparticular. This means that all of the advantages of VLSI processes(e.g., highest level of integration, miniaturization, cost-efficiency,and robustness) can be applied to active-electrode biosensors.Furthermore, the principles of the present invention provide methods bywhich one can design arrays of active-electrode biosensors using CMOSprocesses. While it is self-evident that the present invention can beused for a variety of biosensing applications, embodiments of thepresent invention described herein are related to DNA sequencingapplications.

There are many different techniques to perform DNA sequencing andextract structural information from unknown nucleic acid strands.Certain embodiments of the present invention may rely on theSequence-by-Synthesis (SBS) procedure. In SBS, individual nucleotides(dATP, dCTP, dGTP and dTTP) are iteratively introduced to a primed DNAcomplex in the presence of the DNA polymerase enzyme while theoccurrence of polymerization events is monitored. Successfulpolymerization events at the 3′-terminus of the primer suggest thepresence of the complementary base on the template DNA while theamplitude of the polymerization indicates the number of consecutiveidentical bases. Different techniques have been discussed in the art todetect the polymerization events to perform the SBS procedure. Examplesare bioluminescence-based enzymatic cascade, fluorescent-labelnucleotides, and pH-based. Embodiments of the present inventiondescribed herein provide an alternative electro-analytical techniquewhich is based on using active-electrode biosensors. The key advantagesover the previous methods are amenability to VLSI integration,miniaturization capabilities, lower noise performance, and increaseddetection dynamic range.

DEFINITIONS

An active electrode is defined as a highly conductive material (i.e., anelectrode) with its electrical potential, Φ₀(t), set to V₀(t), a definedvoltage set by an independent time-varying source, such that Φ₀(t)=V₀(t)at all time. In the context of electro-analysis and the presentinvention, an active-electrode system is defined as a conductivematerial (i.e., the electrode) that is capacitively-coupled to anaqueous and electrically-conductive solution (i.e., the electrolyte)such that the capacitively-coupled electrode-to-electrolyte potentialdifference, Φ_(E)(t), follows V₀(t), such that Φ_(E)(t)=V₀(t) at alltime.

An active electrode biosensor is defined here as an active electrodesystem with an electrolyte containing the target analyte in which byapplying time-varying V₀(t) and concurrently monitoring thecorresponding coupled charge (screening charge) on the highly-conductiveelectrode, denoted by Q_(E)(t), one may infer information regarding thetarget analyte presence, and/or abundance and/or molecular structure.

There are unique characteristics for the active electrodes biosensordescribed herein. The first is that “capacitively-coupled” means thatthe active-electrode is not directly in contact with the electrolyte andthat an electrically insulating layer with a thickness between 5 nm to100 nm is placed between the electrode and the electrolyte. The secondis that Q_(E)(t) is located in a very thin layer near theelectrode-insulator interface; however, −Q_(E)(t), the oppositescreening charge on the electrolyte-insulator interface side, isdistributed non-uniformly within the electrolyte in a form generallyreferred to in the art as the double layer (i.e., Helmotz and diffusionlayers). The third and final characteristic is that V₀(t) can changeQ_(E)(t) as well as the profile of the charge within the double layer.It is known in the art that such changes depend on the exactelectrochemical characteristics of the electrolyte (e.g., ionic speciescharges and their diffusion coefficient), the electrolyte-insulatorinterface (e.g., surface pKa and the concentration the surface traps forthe ions in the electrolyte), and the thickness as well as the materialcomposition of the insulator. In view of the foregoing, no net chargetransfer from the electrode to the electrolyte can occur (i.e., no DCcurrent can pass the interface); however, the ionic charges can stillelectrostatically interact with the charge carriers within theelectrode.

Active-Electrode Sensor Circuit Architecture

A simple and widely accepted circuit model 100 for active-electrodesensors is shown in FIG. 1 in accordance with an embodiment of thepresent invention. Referring to FIG. 1, C₁ and C_(D) represent theinsulator layer 101 and double layer 102, respectively, while R_(EL)represents the ohmic resistance of the electrolyte from theelectrode-insulator interface 103 to a counter electrode 104 (e.g.,Ag/AgCl electrode) in the electrolyte. For typical insulating materials,such as SiO₂, Si₃N₄, TiO₂, Al₂O₃ or HfO₂, one can safely assume that C₁is a linear capacitor; however, C_(D) is widely known in the art to beinherently non-linear and function of the voltage placed across it.

The fundamental sensor circuitry 200 in the present invention formeasuring Q_(E)(t) is shown in FIG. 2 in accordance with an embodimentof the present invention which is essentially a Switched-CapacitorCharge Amplifier (SCCA). In this circuit, to ensure Φ_(E)(t)=V₀(t) atall times, one can take advantage of an operational amplifier 201 with acapacitive negative feedback that connects the (−) input of amplifier201 to its output, V_(OUT)(t), while V₀(t) is applied to its (+) input.To detect Q_(E)(t), the following steps occur:

(a) Reset step: First the feedback reset switch is activated (connected)to discharge any accumulated charge on C_(f) at t=0 such thatV_(OUT)(t)=V₀(t).

(b) Read step: Subsequently, at t=Δ, the switch is deactivated(disconnected) and V_(OUT)(t) is read in real time for t>Δ. It can beshown that during this phase

$\begin{matrix}{{V_{OUT}(t)} = {\frac{1}{C_{f}}\left\lbrack {{Q_{E}(t)} - {Q_{E}(\Delta)}} \right\rbrack}} & \left( {{EQ}\mspace{14mu} 1} \right)\end{matrix}$

which means that V_(OUT)(t) is effectively the amplified version of thedifference between the charge at time t compared with the charge at t=Δ.In most cases, the reset step duration (Δ) can be kept relatively smallsuch that Q_(E)(Δ)≈Q_(E)(0), thereby resulting in

$\begin{matrix}{{V_{OUT}(t)} \approx {\frac{1}{C_{f}}\left\lbrack {{Q_{E}(t)} - {Q_{E}(0)}} \right\rbrack}} & \left( {{EQ}\mspace{14mu} 2} \right)\end{matrix}$

which indicate that V_(OUT)(t) is the amplified version differencebetween the charge at time t compared with the charge at t=0.

(c) Iteration steps: Repeat steps (a) and (b) periodically with intervalT, i.e., activating the reset switch at t=T, t=2T, t=3T, . . . anddeactivating it at t=T+Δ, t=2T+Δ, t=3T+Δ, . . . .

(d) Constructing Q_(E)(Δ): Use the individual measured values of eachinterval of step (c) to create the Q_(E)(t)−Q_(E)(0) waveform sampled atthe frequency 1/T.

The minimum detection level of the active-electrode sensor system islimited by the inherent noise sources within SCCA, particularly thenoise contributed by the amplifier. Generally speaking, high-gainamplifiers introduce a high level of low-frequency noise (i.e., 1/fnoise) and DC offset in the system. To suppress both the low-frequencynoise and offset of the active-electrode sensor, one can use differentCorrelated Double Sampling (CDS) techniques which are widely used in theart. In FIG. 3A, an exemplary embodiment of circuitry 300 implementing aCDS technique which requires three additional switches activated bysignals Φ₁, Φ₂, and Φ₃, and an offset storage capacitor, C_(S), is shownin accordance with an embodiment of the present invention. As shown inthe timing diagram 301 of FIG. 3B, initially the charge across the C_(f)is reset and subsequently during the offset calibration phase (high Φ₁and Φ₃), the input referred offset (and noise) of operational amplifier201 is stored on C_(S). Finally, in the readout phase, this storedvoltage is subtracted from the input of operational amplifier 201 (i.e.,the offset and noise are cancelled) and the output becomes independentof this stored value. In this case, Δ is defined as the duration of timebetween the rising edge of the reset and the falling edge of Φ₃.

Active-Electrode Biosensor System

In order to create a biosensor using an active electrode sensor, oneshould devise methods to couple the measurable Q_(E)(t) to thebiosensing interactions that occur between the target analyte and therecognition layer. There are two general approaches to carry this outthis:

Method 1:

The first method is to incorporate the recognition layer within thedouble layer of the active-electrode system, such thatanalyte-recognition layer interactions directly affect the distributionof −Q_(E)(t) and therefore the value of C_(D). A typical exampleapplication for this approach is label-free DNA hybridization detectionin which the capturing DNA strands (in the recognition layer) areattached to the solid surface and are physically immobilized within theinterface double layer of the active-layer. In this example, successfulhybridization of the target charged DNA molecule modifies the interfacecharge and subsequently the C_(D). In FIG. 4, it is illustrated how thistype of biosensor 400 can be accommodated by the SCCA-basedactive-electrode sensor in accordance with an embodiment of the presentinvention. To measure C_(D), which is an indicator ofanalyte-recognition layer interactions, one may apply a sinusoidalsignal 401 at frequency ω across the interface by applying V₀(t)=V₀+V₁cos(ω) (both V₀ and V₁ are constant values) and examine the content ofV_(OUT)(t) at frequency ω. In this case, the phase vector of the output,denoted by V_(OUT)(ω), can be described by the following formula:

$\begin{matrix}{{V_{OUT}(\omega)} = {V_{1}\left\lbrack {1 + {\frac{C_{D}{}C_{I}}{C_{f}} \times \frac{1}{1 + {{j\omega}\; R_{EL}C_{D}{}C_{I}}}}} \right\rbrack}} & \left( {{EQ}\mspace{14mu} 3} \right)\end{matrix}$

Since C_(f), C₁, and V₁ are known values, one can use (EQ 3) and themeasurement at a plurality of frequencies to estimate both C_(D) andR_(EL). By measuring V_(OUT)(ω) at more than two frequencies, redundantinformation is created which can be used to further improve theestimated C_(D) and R_(EL).

Method 2:

The second method is to create ionic currents within the biosensingreaction volume (i.e., coordinated where R_(EL) is the dominantelectrical element) that are indicative of analyte-recognition layerinteractions. An example of such a configuration is common inelectrochemical enzyme biosensors which take advantage of electro-activeenzymes (e.g., horseradish peroxidase or glucose oxidase) attached totheir detection antibody. In FIG. 5, it is illustrated how the activeelectrode sensor 500 can detect such currents in accordance with anembodiment of the present invention. As illustrated in FIG. 5, i(t),represents the ionic current within the solution which is triggered att=0. It is straightforward to show that the output of the SCCA in thiscase, the sensor output for t>0 can be formulated by:

$\begin{matrix}{{V_{OUT}(t)} = {\frac{^{\frac{t}{R_{EL}C_{D}{}C_{I}}}}{C_{f}}{\int_{0}^{t}{^{- \frac{\alpha}{R_{EL}C_{D}{}C_{I}}}{i(\alpha)}\ {\alpha}}}}} & \left( {{EQ}\mspace{14mu} 4} \right)\end{matrix}$

For systems in which i(t) changes occur at a much slower rate comparedto the sensor relaxation time, defined by τ=R_(EL)C_(D)∥C₁, one cansimplify and rewrite (EQ 4) as

$\begin{matrix}{{V_{OUT}(t)} = {\frac{R_{EL}C_{D}{}C_{I}}{C_{f}}{i(t)}}} & \left( {{EQ}\mspace{14mu} 5} \right)\end{matrix}$

One critical issue here is that while (EQ 4) or (EQ 5) offer a means toevaluate i(t) using the SCCA output; however, the exact relationshipbetween these two parameters relies on the values of both R_(EL) andC_(D) which are known to be susceptible to unwanted drifts duringelectro-analysis. This is a known and widely-recognized problem in thisfield. R_(EL) drifts generally happen when the ionic content of theelectrolyte is changed (e.g., by injecting in or washing away differentreagents), or when the reference electrode remains for a long time inthe electrolyte and ages. C_(D) drifts are often a result of unwantedinteraction of the insulator surface with reactants and ions in theelectrolyte which slowly alter the charge distribution at theelectrolyte-insulator surface. If such drifts are not continuallymonitored and effectively calibrated out the quality of the measurementswill be significantly degraded.

Hybrid Method:

In one embodiment, a method for implementing an active electrodebiosensor of the present invention is to concurrently use method 1 andmethod 2 during electro-analysis. The basic idea is to take advantage ofmethod 1 for continual calibration and monitoring of the surface andelectrolyte (C_(D) and/or R_(EL)) and use method 2 to monitor any ioniccurrents. To enable simultaneous operation of both these methods, oneshould operate method 1 in frequencies above the Nyquist bandwidth ofi(t). For example, if the informative frequency content of i(t) iswithin DC to 1 kHz, method 1 is operated in frequencies higher than 1kHz. This approach essentially de-couples the operation of method 1 andmethod 2 by separating their frequency operation.

In FIG. 6, we illustrate an exemplary embodiment of circuitry 600 as tohow the hybrid method can be implemented using the SCCA in accordancewith an embodiment of the present invention. The general idea is to usea Low-Pass filter (LPF) and High-Pass Filter (HPF) to isolate the outputof each method from V_(OUT)(t) and analyze them independently.

Arrays of Active-Electrode Sensors

In some embodiments, an array of active-electrode sensors are built on acommon substrate, such as a semiconductor substrate (e.g., CMOS) and byusing VLSI fabrication processes. In some cases, the number of thepixels within this array is greater than 10 and can be as large as 10⁸per single substrate. Techniques for selecting the row and column of thepixel to be interrogated are widely known to those skilled in the art ofdesign of sensor array and image sensor arrays.

In some situations, a biosensor array includes at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ within across-sectional area of at most about 1000 cm², 100 cm², 10 cm², 1 cm²,0.5 cm², or 0.1 cm².

In some embodiments in which an array of active-electrode sensors isbuilt in a semiconductor substrate, each pixel may have part of therequired circuitry to enable a SSCA-based active electrode sensor, and,for example, the operational amplifier may be shared by a plurality ofpixels in, for example, all the pixels within the column. This method ofsharing the circuitry in the signal path is a widely used method in CMOSsensor array and image sensor arrays. In some embodiments, the sharedcircuits are placed in the periphery of the array to minimize the sizeof the individual pixels.

Detecting DNA Polymerization using Active-Electrode Biosensors

One of the primary applications that is targeted using the principles ofthe present invention is DNA sequencing. There are many differenttechniques to perform DNA sequencing and extract structural informationfrom unknown nucleic acid strands. In some embodiments, DNA sequencingis accomplished via Sequence-by-Synthesis (SBS), in which individualnucleotides (adenine, cytosine, guanine or thymine) are iterativelyintroduced to a primed DNA complex in the presence of the DNA polymeraseenzyme. The occurrence of one or more polymerization events ismonitored, such as with the aid of a biochip described herein.Successful polymerization at the 3′-terminus of the primer is indicativeof the presence of the complementary base on the template DNA while theamplitude of the polymerization is indicative of the number ofconsecutive identical bases. Different approaches have been provided fordetecting the polymerization events and perform SBS. Examples arebioluminescence-based enzymatic cascade (e.g., J. M. Rothberg and J. H.Leamon, “The development and impact of 454 sequencing”, Nature Biotech.,vol. 23, no. 10, pp. 1117-1125, 2008), fluorescent-label nucleotides(e.g., U.S. Pat. No. 7,835,871), and pH-based (e.g., U.S. Pat. No.7,948,015). In the present invention, an alternative electrochemicaltechnique is provided which is not only amendable to integration andminiaturization, but also offers a significantly better noiseperformance and measurement robustness.

In the following, the details of how SBS can be enabled by the activeelectrode biosensors are described. Initially, the electroniccharacteristics of DNA polymerization, where its detection isfundamental in SBS, is discussed followed by discussing the multipleembodiments in which DNA polymerization can be detected using theactive-electrode biosensors.

In DNA polymerization, the 3′-terminus of the primer is extended by theDNA polymerase enzyme which facilitates the incorporation of individualnucleotides (deoxyribonucleotide triphosphates, dNTPs) that arecomplementary to the template DNA strand. A single nucleotideincorporation event that extends the primer from length n to n+1 is bestdescribed by

which states that the DNA-enzyme complex absorbs a single dNTP moleculeand releases a pyrophosphate (PPi) molecule. Since all of theparticipating molecules in the catalytic reaction of (EQ 6) (includingboth the substrates and products) are essentially charged species, it isfeasible to setup certain conditions in which DNA polymerization canresult in a measurable electronic parameter in this system. If this isdone, then monitoring this particular parameter can be used to detect(and quantify) DNA polymerization and therefore can be used to performSBS. All of the DNA sequencing embodiments described herein operateaccording to this particular principle.

Previously, Sakurai et al. (“Real-Time Monitoring of DNA PolymeraseReactions by a Micro ISFET pH Sensor”, Anal. Chem., vol. 64, pp.1996-1997, 1992) demonstrated that a small pH change induced by dNTPincorporation can be detected by a micro ISFET pH sensor and used, inreal-time, to detect DNA polymerization events. Later, Pourmand et al.,(“Direct electrical detection of DNA synthesis”, PNAS, vol. 102, no. 17,pp. 6866-6870, 2006) suggested that the transient electrical signalgenerated by DNA immobilized on a polarized gold electrode duringpolymerization can be sensed in real-time using differential voltage andcurrent amplifiers and be used to sense dNTP incorporations. Recently,Rothberg et al., (“An integrated semiconductor device enablingnon-optical genome sequencing”, Nature, vol. 475, pp. 348-352, 2011)demonstrated how an array of ISFET in conjunction with random beadarrays can be used to create high-throughput parallel pH-based SBSarrays.

In the present invention, an alternative approach is introduced whichuses embodiments of an active-electrode sensor to detect DNApolymerization to enable DNA SBS. The premise of this system is the factthat nucleotide incorporation when the primed DNA is immobilized (i.e.,is attached to a solid surface), can result in an imbalance between theabsorbed and released free ionic charges in the electrolyte near theDNA, which in turn results in localized ionic currents that can besensed by the active-electrode biosensor. Unlike the previously reportedsystem, active-electrode arrays, rely neither on the concentration ofprotons and ISFET structures, nor polarized gold electrodes to detectpolymerization. In addition, active-electrode sensors can also measurethe surface capacitance concurrently with polymerization detection(Hybrid method), a unique feature that none of the aforementionedreferences have.

Referring now to FIG. 7, FIG. 7 is a table 700 illustrating the ionicspecies and the generated net free charge of a single nucleotideincorporation event when DNA is immobilized in accordance with anembodiment of the present invention. As illustrated in FIG. 7, dependingon the pH of the electrolyte and isoelectric characteristics of theinvolved molecules, the generated average net free negative charge (fromPPi) and net free positive charge (from protons, H⁺) are different. Yet,the net generated charge (i.e., the sum of the positive and negativecharges) is always positive and equal to +1. This indicates that DNApolymerization always creates a positive diffusion potential in theelectrolyte at the DNA polymerization coordinate which in turn cancreate an outward going ionic current through free net charge diffusivespreading.

Based on this observation, one can state that if an active-electrodebiosensor is placed in the electrolyte such that it measures the ioniccurrent that is generated by DNA polymerization, one can detect DNApolymerization events. It is important to realize that the DNA itselfdoes not have to be in intimate proximity of the electrode and as longas the current reaches the electrode, one can detect polymerization. Inpractice this means that the electrolyte-insulator surface should bewithin a few diffusion lengths for the PPi and proton ions, which isdefined by the average distance that these ions can move the DNApolymerization until they recombine with other ions in the solution.Depending on the characteristics of the solution (e.g., saltconcentration, buffer capacity and temperature), this distance istypically between 1 nm and 10 nm.

It is noted herein that the embodiments of the present invention arecategorically different from all ISFET-based sensors described in theart. ISFET devices and sensors implemented by them, by their definition,include electrically-floating electrodes where the electrode potentialis undefined (unlike active-electrode sensors which are always set toV_(o)(t)) and can (and should) change during measurements. In addition,unlike Pourmand et. al and its derivatives, active-electrode sensorsdescribed herein do not use clamp or voltage amplifiers and furthermoredo not require the DNA to be attached to the polarized metal electrodesurface to ensure that the DNA backbone protonization is directlyshielded by the electrode.

In some embodiments, the components and procedures to detect DNApolymerization using active electrode biosensors are:

(a) Immobilize a plurality of identical primed DNA molecules in areaction chamber and interface an active-electrode biosensor system toit;

(b) Introduce dNTPs into the reaction chamber in presence of the DNApolymerase enzyme;

(c) Measure in real-time the ionic current, R_(EL), and C_(D) using theaforementioned hybrid biosensing method;

(d) Estimate i(t) by using the measured V_(OUT)(t), R_(EL), and C_(D);and

(e) Use the estimated i(t) to identify the occurrence and amount of DNApolymerization events for the primed DNA molecules interfaced to theactive-electrode biosensor.

In some embodiments, to carryout a DNA SBS procedure and identify thesequence of primed DNA molecules (e.g., a clonal population thereof),the following steps are used:

(a) Immobilize a plurality of identical primed DNA molecules in areaction chamber and interface an active-electrode biosensor system toit;

(b) Introduce a single type of nucleotide (dATP, dTTP, dCTP, or dGTP) inthe presence of DNA polymerase and monitor DNA polymerization;

(c) Remove the remaining unreacted nucleotides from the reactionchamber;

(d) Repeat step (b) through (c) for a different nucleotide; and

(e) Use the occurrence and the quantity of DNA polymerization events tofind the sequence.

In the embodiments of the present invention where the integratedactive-electrode biochip array can carries parallel (multiplexed) DNAsequencing, one has:

(a) An immobilized primed DNA array created on the surface of aCMOS-integrated semiconductor chip where identical DNA strands arelocated in distinct coordinates within the array, referred to herein asthe pixels;

(b) An array of active-electrode biosensors built in a CMOS chip in theform of an integrated circuit in which individual pixels of the DNAarray have one active-electrode biosensor;

(c) A reaction chamber which can contain aqueous solutions on top of theCMOS chip where the DNA array is located;

(d) A fluidic single-directional flow-through system which enablescontrolled injection and removal of different aqueous from the reactionchamber including, but not limited to, dNTPs, DNA polymerase enzyme,wash buffer, dNTPase, and pH standard buffers;

(e) A system to monitor and control the temperature of the reactionchamber; and

(f) A data acquisition and processing device which can extract, read,and process the output of each integrated active electrode biosensor.

Preparing DNA for Sequencing

The nucleic acid being sequenced is referred to herein as the targetnucleic acid. Target nucleic acids include, but are not limited to, DNA,such as but not limited to, genomic DNA, mitochondrial DNA, cDNA and thelike, and RNA, such as but not limited to, mRNA, miRNA, and the like.The nucleic acid may be from any source including naturally occurringsources or synthetic sources. The nucleic acids may be Polymerase ChainReaction (PCR) products, cosmids, plasmids, naturally occurring orsynthetic libraries, and the like. The present invention is not to belimited in this regard. The methods provided herein can be used tosequence nucleic acids of any length. The following is a briefdescription of examples of these methods.

In some embodiments, target nucleic acids are prepared using any mannerknown in the art. As an example, genomic DNA may be harvested from asample according to techniques known in the art (see for exampleSambrook et al. “Maniatis”). Following harvest, the DNA may befragmented to yield nucleic acids of smaller length. The resultingfragments may be on the order of hundreds, thousands, or tens ofthousands nucleotides in length. In some embodiments, the fragments are200-1000 base pairs (bp) in size, or 300-800 by in size, although theyare not so limited. Nucleic acids may be fragmented by any meansincluding but not limited to mechanical, enzymatic or chemical means.Examples include shearing, sonication, nebulization and endonuclease(e.g., Dnase I) digestion, or any other technique known in the art toproduce nucleic acid fragments, optionally of a desired length.Fragmentation can be followed by size selection techniques which can beused to enrich or isolate fragments of a particular length or size. Suchtechniques are also known in the art and include, but are not limitedto, gel electrophoresis or Solid-Phase Reversible Immobilization (SPRI).

In some embodiments, the size selected target nucleic acids are ligatedto adaptor sequences on both the 5′ and 3′ ends. These adaptor sequencescomprise amplification primer sequences to be used in amplifying thetarget nucleic acids. One adaptor sequence may also comprise a sequencecomplementary to the sequencing primer. The opposite adaptor sequencemay comprise a moiety that facilitates binding of the nucleic acid to asolid support, such as but not limited to, a bead. An example of such amoiety is a biotin molecule (or a double biotin moiety, as described byDiehl et al. Nature Methods, 2006, 3(7):551-559) and such a labelednucleic acid can therefore be bound to a solid support having avidin orstreptavidin groups. The resulting nucleic acid is referred to herein asa template nucleic acid. The template nucleic acid comprises at leastthe target nucleic acid and usually comprises nucleotide sequences inaddition to the target.

Immobilization of DNA

There are different known methods in the art that one can use toimmobilize primed DNA strands near an on the active-electrode biosensor.FIGS. 8A-8C illustrates different embodiments for immobilizing clonalDNA in proximity of an active electrode in accordance with an embodimentof the present invention. Referring to FIGS. 8A-8C, FIGS. 8A-8Cillustrate how identical DNA strands 801 are physically placed near theintegrated active biosensors that are embedded in a CMOS chip 802(includes a silicon substrate 809 and metal layers 810). In oneembodiment, DNA strands 801 are immobilized within microwell structures803 covalently using linkers or through base pairing (hybridization) onthe surface of functionalized micro-beads 804, active electrodes 805, orporous polymers 806, referred to as solid support herein. In alternativeembodiments, microwell structure 803 may not be present and DNA strands801 are immobilized covalently using linkers, or through base pairing(hybridization) on the surface of the electrolyte-insulator interface807 (insulator identified as element 808), or porous polymers 806. Thesize of the pixels (parameter X) is preferably between 0.1 μm to 50 μm,while the aspect ratio of individual microwells 803 (i.e., X/Y) variesbetween 0.6 to 3.

In some embodiments, a linker (or spacer) is specifically used todistance the template nucleic acid (and in particular the target nucleicacid sequence comprised therein) from the solid support. This canfacilitate sequencing of the end of the target closest to the surface.Examples of suitable linkers are known in the art (see Diehl et al.Nature Methods, 2006, 3(7):551-559) and include, but are not limited to,carbon-carbon linkers, such as, but not limited to, iSp18.

The beads used in the present invention can be made of any materialincluding, but not limited to, cellulose, cellulose derivatives,gelatin, acrylic resins, glass, silica gels, PolyVinyl Pyrrolidine(PVP), co-polymers of vinyl and acrylamide, polystyrene, polystyrenecross-linked with divinylbenzene or the like (see, MerrifieldBiochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels, dextran,crosslinked dextrans (e.g., Sephadex™), rubber, silicon, plastics,nitrocellulose, natural sponges, metal, and agarose gel (e.g.,Sepharose™). In one embodiment, the beads are streptavidin-coated beads.The bead diameter can depend on the density of the well array used withlarger arrays (and thus smaller sized wells) requiring smaller beads.Generally, the bead size may be about 0.1 μm-10 μm, or 1 μm-5 μm. In anexample, the beads are about 5.91 μm in diameter. In another example,the beads are about 2.8 μm in diameter. It is to be understood that thebeads may or may not be perfectly spherical in shape. It is to beunderstood that other beads may be used and other mechanisms forattaching the nucleic acid to the beads may be utilized.

In some embodiments, a homogeneous population of amplified nucleic acidsis conjugated to one or more beads with the proviso that each bead willultimately be bound to a plurality of identical nucleic acid sequences.The degree of loading of nucleic acid templates onto beads will dependon a number of factors including the bead size and the length of thenucleic acid. In most aspects, maximal loading of the beads is desired.Amplification and conjugation of nucleic acids to solid support, such asbeads, may be accomplished in a number of ways, including, but notlimited to, emulsion PCR as described by Margulies et al. Nature 2005437(15):376-380 and accompanying supplemental materials. In someembodiments, the amplification is a representative amplification. Arepresentative amplification is an amplification that does not alter therelative representation of any nucleic acid species.

Before and/or while in the wells of the flow chamber, the beads areincubated with a sequencing primer that binds to its complementarysequence located on the 3′ end of the template nucleic acid (i.e.,either in the amplification primer sequence or in another adaptorsequence ligated to the 3′ end of the target nucleic acid) and with apolymerase for a time and under conditions that promote hybridization ofthe primer to its complementary sequence and that promote binding of thepolymerase to the template nucleic acid. The primer can be of virtuallyany sequence provided it is long enough to be unique. The hybridizationconditions are such that the primer will hybridize to only its truecomplement on the 3′ end of the template. Suitable conditions aredisclosed in Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

Reaction Temperature

The sequencing reaction can be run at a range of temperatures.Typically, the reaction is run in the range of 30-60° C., 35-55° C., or40-45° C. In some embodiments, it is preferable to run the reaction attemperatures that prevent formation of a secondary structure in thenucleic acid. However, this is balanced with the binding of the primer(and the newly synthesized strand) to the template nucleic acid and thereduced half-life of Pyrophosphatase at higher temperatures. In oneembodiment, a suitable temperature is about 41° C. The solutionsincluding the wash buffers and the dNTP solutions are generally warmedto these temperatures in order not to alter the temperature in thewells. The wash buffer containing Pyrophosphatase, however, ispreferably maintained at a lower temperature in order to extend thehalf-life of the enzyme. Typically, this solution is maintained at about4-15° C., and more preferably at about 4-10° C.

Electrode Fabrication

Referring now to FIGS. 9A-9B, FIGS. 9A-9B illustrate exemplaryCMOS-integrated sensing electrodes 901, 902 in accordance with anembodiment of the present invention. The metal of choice in IntegratedCircuits (ICs) is generally aluminum with certain amount of impurities.Accordingly, in the CMOS-integrated embodiments of the presentinvention, one may use aluminum metal layers 903 as the activeelectrode. The top metal layer in CMOS-integrated sensing electrode 901,902 is the optimal metal layer since it is the closest to the surface ofthe chip which can be coupled to the DNA array. The metal layer inCMOS-integrated sensing electrode 901 is generally covered by a thickpassivation layer (typically made of durable oxides such as SiO₂ andSi₃N₄) to protect the metal chemically and mechanically from theexternal environment. Neither bare aluminum nor aluminum covered by athick dielectric layer is an optimal embodiment for the sensingelectrode of active-electrode biosensors. As a result, an insulatinglayer 904 should be formed. The sensing electrodes 901, 902 of thepresent invention in are formed by first creating openings in thepassivation layer of the CMOS chip 905 (comprised of metal layers 906and silicon substrate 907) and exposing the top metal layer. In oneembodiment, the top metal is subsequently covered by a blanket layer ofan insulator 904 (thickness varies between 5 nm to 2 μm) usingconventional thin-film oxide deposition techniques and the microwells(if any) will be created on its top. Example materials include SiO₂,Ti₃N₄, Si₃N₄, TiO₂, Al₂O₃, and HfO₂. The oxide or nitride layer can beformed by various deposition techniques, such as Chemical VaporDeposition (CVD), Atomic Layer Deposition (ALD), or Physical VaporDeposition (PVD, such as, e.g., sputtering). In alternative embodiments,a noble metal layer 908 (e.g., Pt or Au) is first deposited over theexposed metal by using conventional thin-film metal depositiontechniques (e.g., evaporation or electroplating), and afterwards, theblanket oxide layer is placed on top of it. In one embodiment, thethickness of noble layer 908 can vary between 5 nm to 1 μm.

Integration and SBS Arrays

In a preferred DNA SBS embodiment of the present invention, theactive-electrode biosensor array is built on the semiconductor substrateof a CMOS process fabricated using VLSI fabrication processes. In somecases, the number of the pixels within this array is greater than 10 andcan be as large as 10⁸ per single substrate. Integrated circuit designtechniques for selecting the row and column of the pixel to beinterrogated are widely known to those skilled in the art in the designof CMOS sensor arrays and image sensor arrays.

In some situations, the SBS array includes at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ within across-sectional area of at most about 1000 cm², 100 cm², 10 cm², 1 cm²,0.5 cm², or 0.1 cm².

In some embodiments in which a SBA array is built in a semiconductorsubstrate, each biosensing pixel may have part of the required circuitryto enable a SSCA-based active electrode biosensing, and, for example,the operational amplifier may be shared by a plurality of pixels in, forexample, all the pixels within a column of the array. This method ofsharing the circuitry in the signal path is a widely used method in CMOSsensor arrays and image sensor arrays. In preferred embodiments, theshared circuits are placed in the periphery of the SBS array to minimizethe size of individual biosensing pixels.

Example Embodiment

In this section, it is described herein an exemplary embodiment of thepresent invention. This system has been successfully reduced to practiceusing the 0.18 μm CMOS fabrication process offered by TaiwanSemiconductor Manufacturing Company (TSMC). It is noted for clarity thatthe principles of the preset invention are not to be limited in scope(such as the scope of its applications) to the below described detailsof this embodiment.

Referring now to FIGS. 10A-10C, FIGS. 10A-10C illustrate an integratedactive electrode DNA sequencing biochip and the basic layer structuresof its pixels in accordance with an embodiment of the present invention.Specifically, referring to FIGS. 10A-10C in conjunction with FIGS.8A-8C, FIGS. 10A-10C illustrate the basic structure of theactive-electrode CMOS DNA sequencing biochip and further illustrates thescanning electron microscope image of the CMOS chip surface with andwithout DNA-bead complexes. Each pixel 1001 is 16 μm×16 μm and the totalarray size is 90×90. The electrode (layer M6) 805 is made of aluminumand the insulating layer is aluminum oxide (Al₂O₃). Each pixel 1001 hasa shallow microwell (Y=2 μm) built on its top with a 10 μm×10 μmopenings 1003 to hold a single 10 μm bead 804, on which DNA strands 801are immobilized. In this CMOS process, 6 metal layers (M1-M6) are usedand the capacitors, required for SCCA operation, are created using themetal-insulator-metal (MIM) layers that are offered in this CMOSprocess.

Referring now to FIGS. 11A-11B, FIG. 11A illustrates the generalarchitecture of the CMOS-integrated active-electrode biosensor pixel1101 for DNA sequencing in accordance with an embodiment of the presentinvention and FIG. 11B is a timing diagram 1102 of in-pixel 1101 andout-of-pixel 1103. The goal is to measure i(t) by implementing a SSCAwith CDS to reduce the effect of amplifier 1/f noise and offset. Thenegative feedback of the amplifier keeps the electrode potentialconstant and equal to V₀(t) at all times. The main capacitors in thistopology are the feedback capacitor (C_(F)=90 fF), the offset storingcapacitor (C_(S)=105 fF), and the electrode-electrolyte interfacecapacitor (1 pF<C₁∥C_(D)<10 pF).

FIG. 12 illustrates the transistor-level schematic of the signal chainin accordance with an embodiment of the present invention. Referring nowto FIG. 12, during the calibration phase, the amplifier offset is storedonto C_(S). Subsequently during the readout phase, the voltage stored onC_(S) cancels the offset and low frequency fluctuations while reducingthe amplifier gain error.

In order to minimize the pixel circuitry, only the switches and thedifferential pair (M1 and M2) of the SCCA are integrated in-pixel (pixel(i,j)) 1201 as shown in FIG. 12. The rest of the transistors includingthe tail current source (M3) are shared at the column level 1202(comprised of column analog bus (column (j) analog bus) 1203 and columnamplifier (column (j) amplifier) 1204). Both C_(F) and C_(S) are MIMcapacitors and are placed on top of the active circuitry and below thesensing electrode (see FIGS. 10A-10C). During readout, the i^(th) row ofthe array is activated by ROW[i], which connects the circuitry in thecolumn to the pixel. The outputs of the SCCAs are available at thecolumn level and are multiplexed to provide a single buffered output1205 for the chip. The total consumed power in this chip isapproximately 13 mW using a 3.3V supply.

FIGS. 13A-13C illustrates the electrical performance of the SCCAs inaccordance with an embodiment of the present invention. FIG. 13Aincludes a graph 1301 depicting the frequency versus the input-referrednoise. FIG. 13B includes a graph 1302 depicting the bandwidth versusintegrated noise and FIG. 13C includes a graph 1303 depicting the inputvoltage (V_(IN)) and the output voltage (V_(OUT)). In the frequenciesbelow 10 kHz, the noise power spectral density (PSD) is dominated by 1/fnoise of the operational amplifier which can be suppressed by CDS. Asevident, by using a 5 kHz CDS, the input-referred noise can besuppressed to ˜10 μVrms for a 100 Hz bandwidth. The 1 dB compressionpoint for this amplifier measured at a gain of two is 340 mV whichcorresponds to a detection dynamic range of 90 dB.

FIGS. 14A-14C illustrate the interface capacitance measurements versusthe solution pH in graph 1400 in accordance with an embodiment of thepresent invention. Referring to FIG. 14C, FIG. 14C provides the C_(D)∥C₁measurement as the solution pH is changed. As illustrated in FIG. 14A, a10 mV amplitude sinusoidal voltage with a frequency of 5 kHz is appliedto the reference electrode immersed in the solution. The generatedV_(OUT)(t) (FIG. 14B) is then used to evaluate the SCCA gain andsubsequently C_(D)∥C₁.

FIGS. 15A-15C illustrate the DNA polymerization detection in accordancewith an embodiment of the present invention. Referring now to FIGS.15A-15C, FIG. 15C provides the measurement results of real-time DNApolymerization detection. In this experiment, as shown in FIG. 15A,self-primed biotinylated DNA strands 1501 are immobilized onstreptavidin coated magnetic beads 1502. Deoxynucleotide triphosphates(dATP, dCTP, dGTP, and dTTP nucleotides) 1503 are added sequentially totrigger polymerization and perform SBS as shown in FIG. 15B. As isevident in the experimental results as shown in FIG. 15C, the measuredtransient current is large (in the order of 100s of fA) only when thecorrect nucleotide is added (dTTP 1504 in this case) and negligiblechange can be observed during the control experiment when the unmatchednucleotides (dATTP, dCTP, and dGTP) are introduced into the solution.

FIG. 16 illustrates the micrograph of the active-electrode CMOS biochipfor DNA SBS in accordance with an embodiment of the present invention.As illustrated in FIG. 16, the 90×90 sensor array is placed in themiddle of the chip and the electronic input-output (I/O) of the chip istaken at the periphery. The total chip size is 2.5 mm×2.5 mm.

The experimental protocol for the measurements is:

Reagents:

Biotinylated ss-DNA strand (5′-Biotin-CCTCTGAGTCAAAAAA

AAGCCGTCGTTATACAACGGAACGTTGTATAACGACGGC-3′) from Integrated DNATechnologies (IDT), USA; Streptavidin coated magnetic beads of size 8-10μm from Spherotech, USA; DNA polymerization enzyme used is Klenowfragment in a 10× reaction buffer (500 mM Tris-HCl (pH 8.0 at 25° C.),50 mM MgCl₂, 10 mM DTT) from Fermentas, USA; Deoxynucleotidetriphosphates (dNTP) from Qiagen, USA; Standard buffers.

DNA Hybridization onto the Micro-Beads:

Single stranded biotin-DNA in Tris Buffer (20 mM Tris-HCl buffer, 140 mMNaCl, 20 mM KCl, pH 7.5) was heated at 90° C. for 5 minutes and slowlycooled down to 25° C. at 0.1° C./min. The product was self-primed DNA.

250 μl of 1 mg/50 μl streptavidin-coated magnetic beads was washed with1 mL of 20 mM Tris-Buffer and then dispersed in 250 μl of the samebuffer. Next, 10 nmol of biotin-DNA was added to the solution, followedby incubation on a rotator at room temperature for 2 hours. The excessDNA not bound to the beads was washed away with 500 μl of Tris-bufferfor five times. The highest capacity of the beads for biotin DNA is 0.3nmol/1 mg. At last, the beads were dispersed in 250 μl of 1× Klenowreaction buffer to make a 1 mg/50 μl beads solution.

DNA Polymerization Protocol:

100 μL of magnetic beads (1 mg/50 μL) were deposited in the reservoir ontop of the CMOS chip. 5 μL of Klenow enzyme (5 units/μL) was added. TheAg/AgCl reference electrode was dipped into the reservoir. The CMOS chipwas subsequently activated and the real time data were captured on a PCwhen dNTPs were added one at a time.

Measuring Interface Capacitance vs. pH:

150 μL of Tris-HCl buffer (pH 8.0) was deposited in the reservoir on topof the CMOS chip. The reference electrode (Ag/AgCl) was dipped into thereservoir. Sinusoids of amplitude 10 mV and frequency ranging between1-10 kHz were applied to the reference electrode. The pixel outputamplitude was measured, which was then used to estimate the interfacecapacitance C₁. The pH was changed in steps by adding 1-5 μL quantitiesof 120 mM HCl

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

1-10. (canceled)
 11. A method for detecting DNA polymerization usingactive-electrode biosensors, the method comprising: immobilizing aplurality of primed DNA molecules in a reaction chamber containing anaqueous solution on top of an active-electrode biosensor system;interfacing said active-electrode biosensor system to said plurality ofprimed DNA molecules via an insulating layer, wherein saidactive-electrode biosensor system comprises a sensing electrode, a stackof metal layers disposed adjacent to said sensing electrode and adetection circuitry adjacent to said stack of metal layers, wherein saidstack of metal layers comprises one or more metal layers separated by aninsulating material, wherein said detection circuitry is operativelycoupled to said sensing electrode, wherein said detection circuitryhaving an operational amplifier and a capacitor where said capacitor isin a parallel configuration with respect to said operational amplifier,wherein said detection circuitry further comprises an array ofactive-electrode sensors built on a semiconductor substrate; introducingnucleotides into said reaction chamber in the presence of a DNApolymerase enzyme; measuring an output voltage of said active-electrodebiosensor system; estimating an ionic current using said measured outputvoltage of said active-electrode biosensor system; and identifying anoccurrence and amount of DNA polymerization events associated with saidimmobilized DNA using said estimated ionic current.
 12. The method asrecited in claim 11, wherein said measuring of said output voltage ofsaid active-electrode biosensor system comprises: incorporating ananalyte-recognition layer within a double layer of said active-electrodebiosensor system; and creating ionic currents within a biosensingreaction volume that are indicative of analyte-recognition layerinteractions; wherein said incorporating said analyte-recognition layerwithin said double layer of said active-electrode biosensor system andsaid creating ionic currents within said biosensing reaction volume thatare indicative of said analyte-recognition layer interactions areperformed concurrently.
 13. The method as recited in claim 11 furthercomprising: introducing a single type of nucleotide in the presence ofsaid DNA polymerase enzyme; monitoring said DNA polymerization events inresponse to introducing said single type of nucleotide in the presenceof said DNA polymerase enzyme; and removing remaining nucleotides fromsaid reaction chamber after said introduction of said single type ofnucleotide in the presence of said DNA polymerase enzyme.
 14. The methodas recited in claim 13 further comprising: repeating all previouslyrecited steps for a different nucleotide.
 15. The method as recited inclaim 13, wherein said single type of nucleotide comprises one of thefollowing: dATP, dTTP, dCTP and dGTP.
 16. The method as recited in claim11 further comprising: finding a sequence of primed DNA strands usingsaid identified occurrence and amount of said DNA polymerization events.17. The method as recited in claim 11, wherein said primed DNA moleculesare identical. 18-29. (canceled)